This invention relates to Bacillus subtilis extracytoplasmic function (ECF) "sgr" factors and their function as well as to their encoding and regulatory gene sequences.
The extracytoplasmic function (ECF) subfamily of "sgr" factors are a structurally distinct group of proteins that control a great variety of functions often involving the secretion or uptake of macromolecules or ions in both Grain-negative and Gram-positive bacteria (Lonetto, M. A., Brown, K. L., Rudd, K. E. and Buttner, M. J. (1994) Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial "sgr" factors involved in the regulation of extracytoplasmic functions. Proc Natl Acad Sci USA 91:7573-7577; Missiakas, D. and Raina, S. (1998) The extracytoplasmic function sigma factors: role and regulation. Molecular Microbiology 28:1059-1066). Well-characterized examples include E. coli RpoE ("sgr"E) and FecI and Pseudomonas aeruginosa AlgU. Whole bacterial genome sequencing has revealed numerous new members of this class and suggests the presence of seven "sgr" factors in Bacillus subtilis (Kunst, F., Ogaswara, N., Moszer, I., Albertini, A. M., Alloni, G., Azevedo, A., et al. (1997) The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390:249-256) and ten in Mycobacterium tuberculosis (Cole, S. T., Brosch, R., Parkhill, J., Gamier, T., Churcher, C., Harris, D., et al. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537-544). The roles of the B. subtilis ECF "sgr" factors are largely unexplored, and it is not known whether they control distinct or overlapping sets of genes. None of the ECF "sgr" factors in B. subtilis correspond to known regulatory loci despite extensive genetic analysis of this organism. This suggests that the functions controlled by these proteins are not relevant in standard laboratory culture conditions, that the "sgr" factors are redundant in function, or both.
The roles of two ECF "sgr" factors, "sgr"X and "sgr"W, have been identified by a combination of gene disruption and target promoter identification. It has been found that "sgr"X and "sgr"W activate partially overlapping sets of genes that are optimally transcribed in late logarithmic and early stationary phase, respectively (Huang, X., Fredrick, K. L. and Helmann, J. D. (1998) Promoter recognition by Bacillus subtilis "sgr"W: Autoregulation and partial overlap with the "sgr"X regulon. J Bacteriol 180:3765-3770). One sigX mutant displays a slightly increased sensitivity to heat and oxidative stresses, but has no other obvious phenotypes (Huang, X., Decatur, A., Sorokin, A. and Helmann, J. D. (1997) The Bacillus subtilis "sgr"X protein is an extracytoplasmic function sigma factor contributing to the survival of high temperature stress. J Bacteriol 179:2915-2921).
The fact that many ECF "sgr" factors regulate their own expression can be used to determine the role of each ECF "sgr". The sequence of the autoregulatory promoter site can be used to identify related sequences in the genome that potentially control the transcription of target genes. The sigX operon is preceded by an autoregulatory promoter element, PX, that is exclusively dependent on sigX in vivo (Huang et al., 1997). Like sigX, the sigW gene is autoregulated (Huang et al., 1998). Overproduction of "sgr"W and reconstitution of "sgr"W holoenzyme (E"sgr"W) indicate that E"sgr"W recognizes a subset of "sgr"W-dependent promoters in vitro, including abh, divIC, yrhH and ywbN (Huang et al., 1998). Indeed, each of these four promoters is active in a sigX mutant strain and, for the latter two, the corresponding transcripts are missing in a sigX sigW double mutant (Huang and Helmann, 1998).
Identification of the promoter sequences recognized by "sgr"X and "sgr"W can lead to their use in a variety of assays to identify substances that can modulate bacterial growth and replication.
The sigX operon is preceded by an autoregulatory promoter element, PX, that is exclusively dependent on sigX in vivo (Huang et al., 1997). By saturation mutagenesis, the 10 bases most critical for recognition of PX in vivo have been identified. By using this experimentally derived consensus sequence, 10 putative "sgr"X-dependent promoter elements from the B. subtilis genome have been tested and identified (Huang, X. and Helmann, J. D. (1998) Identification of target promoters for the Bacillus subtilis "sgr"X factor using a consensus-directed search. J Mol Biol 279:165-173).
Of the ten putative "sgr"X-dependent promoters, at least seven are active in vivo and three (csbB, lytR, rapD) are completely dependent on "sgr"X for expression. However, for these three genes, an additional "sgr"X-independent promoter also contributes to expression. Since lytR regulates autolysin expression and csbB encodes a putative membrane bound glycosyl transferase, the "sgr"X regulon may act to modify the cell wall during the transition to a non-growing state (Huang and Helmann, 1998). The four remaining active promoters are partially dependent on the holoenzyme, E"sgr"X, but are also recognized by one or more other forms of holoenzyme (including E"sgr"W). However, since most genes of the sigX regulon can be transcribed by multiple holoenzymes, a sigX mutation is unlikely to abolish gene expression. This may account, in part, for the lack of an obvious phenotype for a sigX mutant.
The Bacillus subtilis sigW gene encodes an extracytoplasmic function (ECF) "sgr" factor that is expressed in early stationary phase from a sigW-dependent autoregulatory promoter, PW. Using a consensus-based search procedure, fifteen operons preceded by promoters similar in sequence to PW were identified. At least fourteen of these promoters are dependent on "sgr"W both in vivo and in vitro as judged by lacZ reporter fusions, run-off transcription assays, and nucleotide resolution start site mapping. Thus, "sgr"W controls a regulon of more than 30 genes, many of which encode membrane proteins of unknown function. The "sgr"W regulon includes a penicillin-binding protein (PBP4*) and a co-transcribed amino acid racemase (RacX), homologs of signal peptide peptidase (YteI), flotillin (YuaG), ABC transporters (YknXYZ), non-heme bromoperoxidase (YdjP), epoxide hydrolase (YfhM), and three small peptides with structural similarities to bacteriocin precursor polypeptides.
Therefore, "sgr"W activates a large stationary phase regulon that functions in detoxification, production of antimicrobial compounds, or both. The "sgr"W regulon and its products can be used to develop expression vector systems that can be used for screening assays to identify new, and potentially efficacious antibacterial agents.